Newton C J, Kantrowitz E R
Department of Chemistry, Boston College, Chestnut Hill, MA 02167.
Proc Natl Acad Sci U S A. 1990 Mar;87(6):2309-13. doi: 10.1073/pnas.87.6.2309.
A recent x-ray structure of aspartate carbamoyltransferase (carbamoyl-phosphate: L-aspartate carbamoyl-transferase, EC 2.1.3.2) with phosphonoacetamide bound [Gouaux, J. E. & Lipscomb, W. N. (1990) Biochemistry 29, 389-402] shows an interaction between Asp-236 of the catalytic chain and Lys-143 of the regulatory chain. Asp-236 is part of the loop containing residues 230-245 (240s) of the catalytic chain that undergoes a significant conformational change between the tight and the relaxed states of the enzyme. Furthermore, side-chain interactions between the 240s loop and other portions of the enzyme have been shown to be important for the low activity and low affinity of the tight state and the high activity and high affinity of the relaxed state. To determine whether the intersubunit link between Lys-143 of the regulatory chain and Asp-236 of the catalytic chain is important for either homotropic cooperativity and/or the heterotropic interactions in aspartate carbamoyltransferase, site-specific mutagenesis was used to replace Asp-236 with alanine. The mutant enzyme exhibits full activity and a loss of both homotropic cooperativity and heterotropic interactions. Furthermore, the aspartate concentration at half the maximal observed specific activity is reduced by approximately 8-fold. The mutant enzyme exhibits normal thermal stability but drastically altered reactivity toward p-hydroxymercuribenzoate. The catalytic subunit of the mutant and wild-type enzymes have very similar properties. These results, in conjunction with previous experiments, suggest that the intersubunit link involving Asp-236 is involved in the stabilization of the 240s loop in its tight-state position and that the regulatory subunits exert their effect on the catalytic subunits by influencing the position of the 240s loop.
近期对结合了膦酰基乙酰胺的天冬氨酸氨甲酰基转移酶（氨甲酰磷酸：L-天冬氨酸氨甲酰基转移酶，EC 2.1.3.2）的X射线晶体结构研究[古奥克斯，J. E. & 利普斯科姆，W. N.（1990年）《生物化学》29卷，389 - 402页]显示，催化链上的天冬氨酸-236与调节链上的赖氨酸-143之间存在相互作用。天冬氨酸-236是催化链中包含230 - 245位（240s环）残基的环的一部分，该环在酶的紧密态和松弛态之间会发生显著的构象变化。此外，240s环与酶的其他部分之间的侧链相互作用已被证明对于紧密态的低活性和低亲和力以及松弛态的高活性和高亲和力很重要。为了确定调节链上的赖氨酸-143与催化链上的天冬氨酸-236之间的亚基间连接对于天冬氨酸氨甲酰基转移酶的同促协同性和/或异促相互作用是否重要，采用位点特异性诱变将天冬氨酸-236替换为丙氨酸。突变酶表现出完全活性，但同时丧失了同促协同性和异促相互作用。此外，最大观察到的比活性一半时的天冬氨酸浓度降低了约8倍。突变酶表现出正常的热稳定性，但对对羟基汞苯甲酸的反应性发生了显著改变。突变酶和野生型酶的催化亚基具有非常相似的性质。这些结果与先前的实验一起表明，涉及天冬氨酸-236的亚基间连接参与了240s环在其紧密态位置的稳定，并且调节亚基通过影响240s环的位置对催化亚基发挥作用。
